Abstract

The ligand selectivity of human (hCRF_2A) and Xenopus laevis (xCRF₂) forms of the corticotropin-releasing factor type 2 (CRF₂) receptor differs. The purpose of this study was to identify amino acids in these two CRF₂receptors conferring these differences. An amino acid triplet in the third extracellular domain (Asp²⁶²Leu²⁶³Val²⁶⁴ in hCRF_2A or Lys²⁶⁴Tyr²⁶⁵Ile²⁶⁶ in xCRF₂) was found to diverge between both receptors. When binding and signaling characteristics of receptor mutants hR2KYI and xR2DLV were assessed, the tri-amino acid motif replacement produced receptors with binding properties resembling the xCRF₂receptor. The converse mutation created a mutant receptor with a binding pharmacology identical to the profile of the hCRF_2Areceptor. This effect was most notable for xR2DLV, which possessed a binding affinity for astressin ∼15-fold greater for astressin than sauvagine. In contrast, the binding profiles of the hCRF_2Areceptor and hR2KYI did not differ. These data indicate that another domain of the xCRF₂ receptor mediated low-affinity binding of astressin. Two amino acids in the first extracellular domain differ in xCRF₂ (Asp⁶⁹Ser⁷⁰) and hCRF_2A (Glu⁶⁶Tyr⁶⁷) receptors. The hCRF_2A receptor mutant (hR2DS-KYI) bound astressin with a low affinity indistinguishable from the xCRF₂ receptor. Therefore, these data demonstrate that ligand selectivity differences between amphibian and human forms of the CRF_2A receptor are governed by these two motifs of the extracellular domains of the xCRF₂ receptor.